Potent Mixed Backbone Antisense Oligonucleotide Safety Suppressed Expression of Mutant C9ORF72 Transcripts and Polypeptides: First in Human Pilot Study

Abstract Expansions of a G4C2 repeat in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two devastating adult-onset neurodegenerative disorders. Proposed disease mechanisms include a gain of toxic functions of the G4C2 repeats, implying that selective reduction in levels of the repeat-containing transcripts would represent a treatment strategy for this disorder. In the present study, using C9-ALS/FTD patient derived cells and C9ORF72 BAC transgenic mice, we have generated and optimized antisense oligonucleotides (ASOs) that selectively blunt expression of G4C2 repeat containing transcripts in both the sense and anti-sense strands of C9ORF72 and effectively suppress tissue levels of polyGP dipeptides. In a single patient harboring mutant C9ORF72 with the G4C2 repeat expressions, repeated dosing by intrathecal delivery of the optimal ASO was well tolerated, leading to significant reductions in levels of CSF polyGP.

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Antisense Oligonucleotide Therapies for Neurodegenerative Diseases

Annual Review of Neuroscience ◽

10.1146/annurev-neuro-070918-050501 ◽

2019 ◽

Vol 42 (1) ◽

pp. 385-406 ◽

Cited By ~ 41

Author(s):

C. Frank Bennett ◽

Adrian R. Krainer ◽

Don W. Cleveland

Keyword(s):

Neurodegenerative Diseases ◽

Spinal Muscular Atrophy ◽

Antisense Oligonucleotide ◽

Antisense Oligonucleotides ◽

Muscular Atrophy ◽

Neurological Diseases ◽

Great Promise ◽

Disease Symptoms ◽

Therapeutic Platform ◽

Lateral Sclerosis

Antisense oligonucleotides represent a novel therapeutic platform for the discovery of medicines that have the potential to treat most neurodegenerative diseases. Antisense drugs are currently in development for the treatment of amyotrophic lateral sclerosis, Huntington's disease, and Alzheimer's disease, and multiple research programs are underway for additional neurodegenerative diseases. One antisense drug, nusinersen, has been approved for the treatment of spinal muscular atrophy. Importantly, nusinersen improves disease symptoms when administered to symptomatic patients rather than just slowing the progression of the disease. In addition to the benefit to spinal muscular atrophy patients, there are discoveries from nusinersen that can be applied to other neurological diseases, including method of delivery, doses, tolerability of intrathecally delivered antisense drugs, and the biodistribution of intrathecal dosed antisense drugs. Based in part on the early success of nusinersen, antisense drugs hold great promise as a therapeutic platform for the treatment of neurological diseases.

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Antisense oligonucleotides

Neurology Genetics ◽

10.1212/nxg.0000000000000323 ◽

2019 ◽

Vol 5 (2) ◽

pp. e323 ◽

Cited By ~ 34

Author(s):

Daniel R. Scoles ◽

Eric V. Minikel ◽

Stefan M. Pulst

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Neurodegenerative Diseases ◽

Antisense Oligonucleotide ◽

Antisense Oligonucleotides ◽

Muscular Atrophy ◽

Target Sequence ◽

Spinocerebellar Ataxias ◽

Complementary Oligonucleotide ◽

Disease Modifying ◽

Lateral Sclerosis

There are few disease-modifying therapeutics for neurodegenerative diseases, but successes on the development of antisense oligonucleotide (ASO) therapeutics for spinal muscular atrophy and Duchenne muscular dystrophy predict a robust future for ASOs in medicine. Indeed, existing pipelines for the development of ASO therapies for spinocerebellar ataxias, Huntington disease, Alzheimer disease, amyotrophic lateral sclerosis, Parkinson disease, and others, and increased focus by the pharmaceutical industry on ASO development, strengthen the outlook for using ASOs for neurodegenerative diseases. Perhaps the most significant advantage to ASO therapeutics over other small molecule approaches is that acquisition of the target sequence provides immediate knowledge of putative complementary oligonucleotide therapeutics. In this review, we describe the various types of ASOs, how they are used therapeutically, and the present efforts to develop new ASO therapies that will contribute to a forthcoming toolkit for treating multiple neurodegenerative diseases.

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Amyotrophic Lateral Sclerosis (ALS): Disease Mechanisms☆

Reference Module in Neuroscience and Biobehavioral Psychology ◽

10.1016/b978-0-12-809324-5.02068-x ◽

2017 ◽

Author(s):

C. Vande Velde

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Disease Mechanisms ◽

Lateral Sclerosis

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Phase separation and toxicity of C9orf72 poly(PR) depends on alternate distribution of arginine

The Journal of Cell Biology ◽

10.1083/jcb.202103160 ◽

2021 ◽

Vol 220 (11) ◽

Author(s):

Chen Chen ◽

Yoshiaki Yamanaka ◽

Koji Ueda ◽

Peiying Li ◽

Tamami Miyagi ◽

...

Keyword(s):

Phase Separation ◽

Charge Distribution ◽

Protein Interactions ◽

Protein Protein Interactions ◽

C9orf72 Gene ◽

And Diffusion ◽

Lateral Sclerosis ◽

Dipeptide Repeat ◽

Liquid Liquid Phase Separation

Arg (R)-rich dipeptide repeat proteins (DPRs; poly(PR): Pro-Arg and poly(GR): Gly-Arg), encoded by a hexanucleotide expansion in the C9ORF72 gene, induce neurodegeneration in amyotrophic lateral sclerosis (ALS). Although R-rich DPRs undergo liquid–liquid phase separation (LLPS), which affects multiple biological processes, mechanisms underlying LLPS of DPRs remain elusive. Here, using in silico, in vitro, and in cellulo methods, we determined that the distribution of charged Arg residues regulates the complex coacervation with anionic peptides and nucleic acids. Proteomic analyses revealed that alternate Arg distribution in poly(PR) facilitates entrapment of proteins with acidic motifs via LLPS. Transcription, translation, and diffusion of nucleolar nucleophosmin (NPM1) were impaired by poly(PR) with an alternate charge distribution but not by poly(PR) variants with a consecutive charge distribution. We propose that the pathogenicity of R-rich DPRs is mediated by disturbance of proteins through entrapment in the phase-separated droplets via sequence-controlled multivalent protein–protein interactions.

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Motor Neuron Diseases

10.1093/med/9780197512166.003.0086 ◽

2021 ◽

pp. 752-759

Author(s):

Eric J. Sorenson

Keyword(s):

Motor Neuron ◽

Incidence Rate ◽

Motor Neurons ◽

Anterior Horn Cell ◽

Adult Onset ◽

Motor Neuron Diseases ◽

Kii Peninsula ◽

Geographic Boundaries ◽

Spinal Muscular Atrophies ◽

Lateral Sclerosis

The motor neuron disorders are a clinically diverse group of diseases that share a pathologic loss of the motor neurons. The most common adult-onset disorder is amyotrophic lateral sclerosis (ALS). Other forms include the spinal muscular atrophies, infectious motor neuronopathies, and rare focal forms of anterior horn cell loss.Overall, the incidence rate of ALS is believed to be 1.5 to 2.0 cases per 100,000 person-years, and the prevalence rate is 4 to 6 cases per 100,000 population. Other than in sparsely populated geographic clusters (eg, Guam and the Kii Peninsula of Japan), the incidence rate seems consistent across ethnic and geographic boundaries.

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C9orf72 Gene Expression in Frontotemporal Dementia and Amyotrophic Lateral Sclerosis

Bulletin of Experimental Biology and Medicine ◽

10.1007/s10517-020-04952-0 ◽

2020 ◽

Vol 169 (5) ◽

pp. 673-676

Author(s):

Yu. A. Shpilyukova ◽

E. Yu. Fedotova ◽

N. Yu. Abramycheva ◽

I. A. Kochergin ◽

I. V. Zakroyshchikova ◽

...

Keyword(s):

Gene Expression ◽

Amyotrophic Lateral Sclerosis ◽

Frontotemporal Dementia ◽

C9orf72 Gene ◽

Lateral Sclerosis

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Regulation of PAI-1 Concentration in Platelets by Systemic Administration of Antisense Oligonucleotides to Rats

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615968 ◽

2001 ◽

Vol 85 (06) ◽

pp. 1086-1089 ◽

Cited By ~ 3

Author(s):

Zofia Pawlowska ◽

Ewa Chabielska ◽

Anna Kobylanska ◽

Anna Maciaszek ◽

Maria Swiatkowska ◽

...

Keyword(s):

Antisense Oligonucleotide ◽

Antisense Oligonucleotides ◽

Low Doses ◽

Significant Delay ◽

Occlusion Time ◽

Antisense Approach ◽

First Time ◽

Pai 1 ◽

Arterial Thrombolysis

SummaryIn this report we tested the effect of oligodeoxyribonucleotides antisense to PAI-1 mRNA administered into rats on PAI-1 concentration in platelets. Low doses of the antisense oligonucleotide (MPO-16R) reduced PAI-1 activity, both in rat blood plasma and platelet lysates by 20.5% and 28.7%, respectively. There was no change in platelet count after treatment with MPO-16R but treated platelets showed lower aggregability as compared with controls (37 ± 13% and 54 ± 12%, respectively). In an experimental model of rat arterial thrombosis, low doses of MPO-16R caused a significant delay in the occlusion time (31.8%). These data further support for the role of PAI-1 as a major determinant of arterial thrombolysis resistance and for the first time demonstrate the possibility of reduction of platelet PAI-1 concentration by antisense approach.

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Rapid effect on suckling of an oxytocin antisense oligonucleotide administered into rat supraoptic nucleus

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1994.267.3.r852 ◽

1994 ◽

Vol 267 (3) ◽

pp. R852-R858 ◽

Cited By ~ 6

Author(s):

I. Neumann ◽

D. W. Porter ◽

R. Landgraf ◽

Q. J. Pittman

Keyword(s):

Weight Gain ◽

Antisense Oligonucleotide ◽

Antisense Oligonucleotides ◽

Supraoptic Nucleus ◽

Milk Ejection ◽

Specific Effects ◽

Rapid Effect ◽

Left And Right ◽

Time To Onset ◽

Neuroendocrine Functions

Suckling in the lactating rat (days 6-10 of lactation) was used to characterize the effects of intracerebral oxytocin (Oxt) antisense oligonucleotide treatment on the hypothalamo-neurohypophysial system. Vehicle, mixed bases, Oxt antisense, or vasopressin antisense oligonucleotides (2.5 micrograms/0.5 microliter each) were infused directly into the left and right supraoptic nucleus (SON), 4 h before a litter of 10 pups was allowed to suckle for 30 min. In the Oxt antisense group, there was a significant reduction in the number of milk ejection reflexes (to 34.6 +/- 4.88%, P < 0.001), as well as in the weight gain of the litter (to 18.8 +/- 6.98%, P < 0.03) compared with vehicle (100%)-, mixed base-, or vasopressin antisense-treated animals, which did not differ from each other. The time to onset of suckling, however, was unchanged. Compared with presuckling values, plasma Oxt was significantly increased in all four groups 30 min after onset of suckling (vehicle, to 325 +/- 117%; mixed bases, to 258 +/- 48.2%; vasopressin antisense, to 330 +/- 55.7%), but this increase was less pronounced in Oxt antisense-treated rats (to 157 +/- 20.5%; P < 0.05 vs. vasopressin antisense). In contrast to these changes in neuroendocrine functions during suckling, Oxt as well as vasopressin content and Oxt immunoreactivity in posterior pituitaries and the SON, respectively, did not differ among groups. Our data indicate rapid and specific effects of an Oxt antisense oligonucleotide infused into the SON on neuroendocrine, suckling-related parameters that are not due to depleted stores of Oxt in the hypothalamo-neurohypophysial system.

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Effect of antisense oligonucleotides on the expression of hepatocellular bile acid and organic anion uptake systems in Xenopus laevis oocytes

Biochemical Journal ◽

10.1042/bj3160901 ◽

1996 ◽

Vol 316 (3) ◽

pp. 901-904 ◽

Cited By ~ 82

Author(s):

Bruno HAGENBUCH ◽

Bruce F. SCHARSCHMIDT ◽

Peter J. MEIER

Keyword(s):

Bile Acid ◽

Xenopus Laevis ◽

Rat Liver ◽

Antisense Oligonucleotide ◽

Antisense Oligonucleotides ◽

Organic Anion ◽

Xenopus Laevis Oocytes ◽

Acid Uptake ◽

Uptake System ◽

Anion Uptake

A Na+-dependent bile acid (Na+/taurocholate co-transporting polypeptide; Ntcp) and a Na+-independent bromosulphophthalein (BSP)/bile acid uptake system (organic-anion-transporting polypeptide; oatp) have been cloned from rat liver by using functional expression cloning in Xenopus laevis oocytes. To evaluate the extent to which these cloned transporters could account for overall hepatic bile acid and BSP uptake, we used antisense oligonucleotides to inhibit the expression of Ntcp and oatp in Xenopus laevis oocytes injected with total rat liver mRNA. An Ntcp-specific antisense oligonucleotide co-injected with total rat liver mRNA blocked the expression of Na+-dependent taurocholate uptake by approx. 95%. In contrast, an oatp-specific antisense oligonucleotide when co-injected with total rat liver mRNA had no effect on the expression of Na+-dependent taurocholate uptake, but it blocked Na+-independent uptake of taurocholate by approx. 80% and of BSP by 50%. Assuming similar expression of hepatocellular bile acid and organic anion transporters in Xenopus laevis oocytes, these results indicate that Ntcp and oatp respectively represent the major, if not the only, Na+-dependent and Na+-independent taurocholate uptake systems in rat liver. By contrast, the cloned oatp accounts for only half of BSP transport, suggesting that there must be additional, non-bile acid transporting organic anion uptake systems in rat liver.

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